Effects of SGLT2 inhibitor dapagliflozin in patients with type 2 diabetes on skeletal muscle cellular metabolism

Yvo J M Op den Kamp, Anne Gemmink, Marlies de Ligt, Bas Dautzenberg, Esther Kornips, Johanna A Jorgensen, Gert Schaart, Russell Esterline, Diego A Pava, Joris Hoeks, Vera B Schrauwen-Hinderling, Sander Kersten, Bas Havekes, Timothy R Koves, Deborah M Muoio, Matthijs K C Hesselink, Jan Oscarsson, Esther Phielix, Patrick Schrauwen, Yvo J M Op den Kamp, Anne Gemmink, Marlies de Ligt, Bas Dautzenberg, Esther Kornips, Johanna A Jorgensen, Gert Schaart, Russell Esterline, Diego A Pava, Joris Hoeks, Vera B Schrauwen-Hinderling, Sander Kersten, Bas Havekes, Timothy R Koves, Deborah M Muoio, Matthijs K C Hesselink, Jan Oscarsson, Esther Phielix, Patrick Schrauwen

Abstract

Objective: SGLT2 inhibitors increase urinary glucose excretion and have beneficial effects on cardiovascular and renal outcomes; the underlying mechanism may be metabolic adaptations due to urinary glucose loss. Here, we investigated the cellular and molecular effects of 5 weeks of dapagliflozin treatment on skeletal muscle metabolism in type 2 diabetes patients.

Methods: Twenty-six type 2 diabetes mellitus patients were randomized to a 5-week double-blind, cross-over study with 6-8-week wash-out. Skeletal muscle acetylcarnitine levels, intramyocellular lipid (IMCL) content and phosphocreatine (PCr) recovery rate were measured by magnetic resonance spectroscopy (MRS). Ex vivo mitochondrial respiration was measured in skeletal muscle fibers using high resolution respirometry. Intramyocellular lipid droplet and mitochondrial network dynamics were investigated using confocal microscopy. Skeletal muscle levels of acylcarnitines, amino acids and TCA cycle intermediates were measured. Expression of genes involved in fatty acid metabolism were investigated.

Results: Mitochondrial function, mitochondrial network integrity and citrate synthase and carnitine acetyltransferase activities in skeletal muscle were unaltered after dapagliflozin treatment. Dapagliflozin treatment increased intramyocellular lipid content (0.060 (0.011, 0.110) %, p = 0.019). Myocellular lipid droplets increased in size (0.03 μm2 (0.01-0.06), p < 0.05) and number (0.003 μm-2 (-0.001-0.007), p = 0.09) upon dapagliflozin treatment. CPT1A, CPT1B and malonyl CoA-decarboxylase mRNA expression was increased by dapagliflozin. Fasting acylcarnitine species and C4-OH carnitine levels (0.4704 (0.1246, 0.8162) pmoles∗mg tissue-1, p < 0.001) in skeletal muscle were higher after dapagliflozin treatment, while acetylcarnitine levels were lower (-40.0774 (-64.4766, -15.6782) pmoles∗mg tissue-1, p < 0.001). Fasting levels of several amino acids, succinate, alpha-ketoglutarate and lactate in skeletal muscle were significantly lower after dapagliflozin treatment.

Conclusion: Dapagliflozin treatment for 5 weeks leads to adaptive changes in skeletal muscle substrate metabolism favoring metabolism of fatty acid and ketone bodies and reduced glycolytic flux. The trial is registered with ClinicalTrials.gov, number NCT03338855.

Keywords: Acylcarnitines; Dapagliflozin; Mitochondrial function; Myocellular lipid metabolism; SGLT2i; TCA cycle Intermediates.

Copyright © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
Effects of dapagliflozin on mitochondrial function and acetylcarnitine levels.(A) Phosphocreatine (PCr) recovery rate (n = 22), (B)ex vivo mitochondrial respiration from vastus lasteralis muscle biopsies taken after an overnight fast (n = 22), (C) Citrate synthase activity (n = 21), (D) average acetylcarnitine levels at rest (n = 21), maximal acetylcarnitine levels after exercise (n = 21), and average acetylcarnitine levels after exercise (n = 21), and (E) plasma lactate levels during exercise (n = 20) after placebo (P) and dapagliflozin (D) treatment. Placebo condition = white bars, dapagliflozin condition = grey bars. Results are given as least squares mean (LSM) and 95% confidence interval (CI), obtained through a linear mixed model. ∗P < 0.05 vs. placebo by Wilcoxon paired signed-rank test.
Figure 2
Figure 2
Increased intramyocellular lipid content after dapagliflozin treatment.(A) intramyocellular lipid (IMCL) content of m. tibialis anterior measured with 1H-MRS and expressed as CH2 intensity relative to water resonance (%), (B) Pearson correlation between change in insulin sensitivity (delta RDhigh-basal) and change in IMCL, and (C) spearman correlation between change in whole-body lipid oxidation and change in skeletal muscle acetylcarnitine (C2 carnitine) levels. Placebo condition = white bars, dapagliflozin condition = grey bars. Results (n = 20) are given as least squares mean (LSM) and 95% confidence interval (CI), obtained through a linear mixed model. ∗P < 0.05 vs. placebo by Wilcoxon paired signed-rank test.
Figure 3
Figure 3
Effects of dapagliflozin on Intramyocellular lipid droplet morphology in vastus lateralis.(A) representative images of LDs stained in green and cell membranes stained in red of type 1 and type 2 muscle fibers after placebo and dapagliflozin treatment. (B)–(D) quantification of lipid area fraction, LD number and LD size respectively. Placebo condition = white bars, dapagliflozin condition = grey bars. Results (n = 10) are in least squares mean (LSM) and 95% confidence interval (CI), obtained through a linear mixed model. ∗P < 0.05 vs. placebo by Wilcoxon paired signed-rank test. Muscle biopsies were taken in the overnight fasted state.
Figure 4
Figure 4
Effects of dapagliflozin on fasting acylcarnitine and CrAT activity.(A) Fold change in skeletal muscle levels of carnitine species after an overnight fast (n = 22), and (B) creatine acetyltransferase activity (n = 22). Results in b are in least squares mean (LSM) and 95% confidence interval (CI), obtained through a linear mixed model.
Figure 5
Figure 5
Effects of dapagliflozin on expression of genes involved in fatty acid metabolism. Fold change in expression of genes in skeletal muscle fatty acid metabolism (n = 22).
Figure 6
Figure 6
Amino acids and TCA cycle intermediates levels.(A) Fold change in skeletal muscle levels of amino acids after an overnight fast (n = 22), and (B) fold change in skeletal muscle levels of TCA cycle intermediates (n = 22).

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